Protein C in its activated form plays an important role in regulating blood coagulation. The activated protein C, a serine protease, inactivates coagulation Factors Va and VIIIa by limited proteolysis. The coagulation cascade initiated by tissue injury, for example, is prevented from proceeding in an unimpeded chain-reaction beyond the area of injury by protein C.
Protein C is synthesized in the liver as a single chain precursor polypeptide which is subsequently processed to a light chain of about 155 amino acids (M.sub.r =21,000) and a heavy chain of 262 amino acids (M.sub.r =40,000). The heavy and light chains circulate in the blood as a two-chain inactive protein, or zymogen, held together by a disulfide bond. When a 12 amino acid residue is cleaved from the amino-terminus of the heavy chain portion of the zymogen in a reaction mediated by thrombin, the protein becomes activated. Another blood protein, referred to as "protein S," is believed to somehow accelerate the protein C-catalyzed proteolysis of Factor Va.
Protein C has also been implicated in the action of tissue-type plasminogen activator (Kisiel and Fujikawa, Behring Inst, Mitt. 73:29-42, 1983). Infusion of bovine activated protein C (APC) into dogs results in increased plasminogen activator activity (Comp and Esmon, J. Clin. Invest. 68:1221-1228, 1981). Other studies (Sakata et al., Proc. Natl. Acad. Sci. USA 82:1121-1125, 1985) have shown that addition of APC to cultured endothelial cells leads to a rapid, dose-dependent increase in fibrinolytic activity in the conditioned media, reflecting increases in the activity of both urokinase-related and tissue-type plasminogen activators. APC treatment also results in a dose-dependent decrease in anti-activator activity. In addition, studies with monoclonal antibodies against endogenous APC (Snow et al. FASEB Abstracts, 1988) implicate APC in maintaining patency of arteries during fibrinolysis and limiting the extent of tissue infarct.
Experimental evidence indicates that activated protein C may be clinically useful in the treatment of thrombosis. Several studies with baboon models of thrombosis have indicated that APC in low doses will be effective in prevention of fibrin deposition, platelet deposition and loss of circulation (Gruber et al., Hemostasis and Thrombosis 374a: abstract 1353, 1987; Widrow et al., Fibrinolysis 2 suppl. 1: abstract 7, 1988; Griffin et al., Thromb. Haemostasis 62: abstract 1512, 1989). The use of APC bypasses the need for in vivo activation of protein C, thus providing a faster acting therapeutic agent.
In addition, exogenous activated protein C has been shown to prevent the coagulopathic and lethal effects of gram negative septicemia (Taylor et al., J. Clin. Invest. 19:918-925, 1987). Data obtained from studies with baboons suggest that activated protein C plays a natural role in protecting against septicemia.
Protein C may be purified from clotting factor concentrates (Marlar et al., Blood 59:1067-1072, 1982) or from plasma (Kisiel, J. Clin. Invest. 64:761-769, 1979) and activated in vitro, but the resulting product may be contaminated with such infectious agents as hepatitis virus, cytomegalovirus, or human immunodeficiency virus (HIV).
More recently, methods for producing activated protein C through recombinant DNA technology have been described. Foster et al. (published European Patent Application EP 215,548) disclose the production of activated protein C through the use of cultured mammalian cells transfected with a protein C DNA sequence from which the coding sequence for the activation peptide has been deleted. Foster et al. (EP 266,190) disclose the production of recombinant activated protein C using a DNA sequence encoding an APC precursor with a modified cleavage site.
Moreover, native human activated protein C (either plasma-derived or recombinant) has a relatively short half-life when administered in vivo (about twenty minutes), necessitating the inconvenience of large doses or frequent administration.
Despite the advances in activated protein C production made possible by the use of genetic engineering, yields remain low and the protein is subject to degradation and/or inactivation during the production process. Thus, there remains a need in the art for methods that enable the production of active activated protein C at higher levels and especially the production of molecules which have a substantially increased half-life in vivo. Quite surprisingly, the present invention fulfills these and other related needs.